Encapsulated kidney tissue

ABSTRACT

Provided are therapeutic implants comprising renal tissue encapsulated within a polymer bead. Also disclosed are methods for treating a disease state in a subject comprising implanting within said subject a therapeutic implant comprising renal tissue encapsulated within a polymer bead. Also provided are methods for making a therapeutic implant comprising: providing renal tissue; mixing the renal tissue with a solution comprising a polymer, thereby forming a tissue-polymer suspension; extruding the tissue-polymer suspension into an bead-forming solution, thereby forming a therapeutic implant comprising beads of said polymer within which the renal tissue is encapsulated.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit to U.S. Provisional Patent ApplicationNo. 61/015,328, filed Dec. 20, 2007, the contents of which areincorporated by reference herein in their entirety.

FIELD OF THE INVENTION

The present invention pertains to the encapsulation of tissue inpolymer.

BACKGROUND OF THE INVENTION

The kidney plays a critical role in maintaining physiologicalhomeostasis. Among its homeostatic functions are acid-base balance,regulation of electrolyte concentrations, blood pressure and bloodvolume regulation. The kidneys accomplish these functions independently,as well as through coordination with other organ systems through theactions of hormones and proteins secreted into the bloodstream. Thesesecreted proteins include erythropoietin (Epo), urodilatin, renin andvitamin D, as well as less emphasized proteins such as adiponectin andleptin.

Adiponectin (also known as AdipoQ, Acrp30, apM1, and GBP28) is anadipocyte-derived cytokine that has been shown to have anti-inflammatoryproperties. In addition, it functions to regulate blood glucose levelsvia cross-communication with the liver. Normal blood concentrations ofadiponectin are 5-30 μg/ml in humans. Previous studies have shown thatcirculating levels of adiponectin are elevated during chronic caloricrestriction in both humans and mice. In contrast, low levels ofadiponectin in human plasma correlate with high insulin, glucose, andtriglycerides, as well as increased obesity. It has been shown thatover-expression of human adiponectin in transgenic mice resulted insuppression of fat accumulation and prevention of premature death by ahigh-calorie diet. Furthermore, a diabetes susceptibility locus has beenmapped to chromosome 3q27, the location of the human adiponectin gene.Increasing adiponectin blood levels could have therapeutic value intreating diabetes and related comorbidities.

Leptin is a 16-kilodalton-protein hormone that plays a key role inregulating energy intake and energy expenditure by decreasing appetiteand increasing metabolism. Recently, leptin has been shown to play arole in protecting the kidneys from renal injury in a mouse model ofdiabetic nephropathy. In addition, leptin promotes angiogenesis byup-regulating vascular endothelial growth factor.

For over thirty years, erythropoietin (Epo), a 30.4 kDa proteinsynthesized and secreted mainly by the kidney, has been successfullyused to stimulate erythropoiesis in patients suffering from anemia.Recently, it has become apparent that the beneficial effects of EPOextend well beyond the stimulation of red blood cell production (Brineset al., Kidney Int., 2006; 70(2):246-250). Previous studies by Chong etal. established that Epo protects the vascular endothelium againstischemic injury. Chong et al., Circulation, 2002; 106 (23):2973-9.Others have confirmed these findings, demonstrating that Epo has aprotective effect on endothelial cells in diverse animal models ofvascular disease (Santhanam et al., Stroke, 2005; 36 (12):2731-7; Satohet al., Circulation, 2006; 113(11):1442-50; Urao et al., CirculationResearch, 2006; 98(11):1405-13). In chronic renal failure, patientsdevelop anemia due to inadequate Epo production by the kidney.Recombinant Epo, administered as a replacement therapy, restoreshematocrit and blood hemoglobin concentrations, eliminating the need forblood transfusions. This treatment, however, entails regular injectionsof Epo, two to four times per week, given either intravenously orsubcutaneously. Epo dosing is cumbersome, resulting in patientnon-compliance and frequent, cyclical fluctuations in blood Epo andhematocrit values.

In light of the protective effects of Epo on the cardiovascular system,as well as the current challenges associated with recombinant Epotreatment, implantation of an Epo-eluting device may be an effectivealternative to the current treatment modality. Such an implantabledevice might also better control hematocrit values and potentially evenprotect organ microvasculature from injury.

Recent studies focused on developing alternative Epo delivery systemsare in progress. Investigators have demonstrated the feasibility ofencapsulating recombinant Epo in different types of bioabsorbablepolymers (See, e.g., Yeh et al., J Microencapsulation, 2007;24(1):82-93; Pistel et al., J. of Controlled Release, 1999;59(3):309-325; Bittner et al., European Journal of Pharmaceutics anBiopharmaceutics, 1998; 45:295-305; Morlock et al., Journal ofControlled Release, 1998; 56:105-115). While encapsulation of peptidesand small molecules into biodegradable envelopes can be achieved usingseveral techniques, the encapsulation of proteins has associatedchallenges. For example, it has been difficult to obtain continuous Eporelease profiles with minimal initial burst as well as sufficientprotein loading within the microspheres. The development of arecombinant Epo-loaded, implantable device may require frequentdrug-reloading or device replacement to ensure long-term, robust diseasemanagement.

Other investigators have placed less emphasis on recombinant Epo and arepursuing a genetic engineering and cell therapy approach. Naffakah etal. examined whether the secretion of Epo from genetically modifiedcells could represent an alternative to repeated injections for treatingchronic anemia. Naffakh et al., Human Gene Therapy, 1996; 7(1):11-21. Inthis study, primary mouse skin fibroblasts were transduced with aretroviral vector in which the murine Epo cDNA was expressed under thecontrol of the murine phosphoglycerate kinase promoter. These“Neo-organs” containing the genetically modified fibroblasts embeddedinto collagen gels were implanted into the peritoneal cavity of miceresulting in an increase in hematocrit and serum Epo concentrationsafter a 10-month observation period. The implantation of Epo-secretingfibroblasts represents a potential method for permanent in vivo Epodelivery.

Similarly, Orive et al. investigated the long-term functionality of anex vivo gene therapy approach. Orive G et al., Molecular Therapy, 2005;12(2):283-9. Polymer microcapsules loaded with Epo-secreting myoblastswere implanted into the peritoneum and subcutaneous tissue of syngeneicand allogeneic mice. High and constant hematocrit levels were maintainedfor more than 100 days in all implanted mice. Interestingly, thefunctionality of capsules implanted in the allogeneic mice persisteduntil day 210 after implantation. These results demonstrate thefeasibility of cell encapsulation technology for the long-term deliveryof Epo within an allogenic model.

In addition, many companies are also developing cell encapsulationtechnology. StemCells (CytoTherapeutics) is developing cell capsulesthat can be surgically implanted and release substances that cross theblood-brain barrier for neurological applications. Novocell Inc. (SanDiego, Calif.) is developing encapsulated islet cells forinsulin-dependent diabetes. Islet Technology, Inc. (St. Paul, Minn.) isalso developing islet microencapsulation technology and has demonstratedthe long-term persistence of their implants in a diabetic dog for morethan 3 years. Amcyte Inc. (Santa Monica, Calif.) is developing isletcells to form an artificial pancreas using photocross-linkable alginateor polyethylene glycol capsule. Finally, MicroIslet Inc. (San Diego,Calif.) is developing a suspension of microencapsulated, porcine isletsfor injection into the abdominal cavity using a highly biocompatiblealginate.

Indeed, several efforts exist, attempting to exploit cell and proteinencapsulation as a means to deliver therapeutic agents. In total, thestate-of-the-art has generated very compelling and useful data, andthese efforts have demonstrated the utility of encapsulation as a methodfor the controlled, long-term delivery of Epo in vivo. However, thereare considerable safety issues that must be resolved before theencapsulation of genetically modified cells can be utilized fortherapeutic proposes.

There remains a need for implantable devices that overcome traditionalproblems associated with therapeutic deployment.

SUMMARY OF THE INVENTION

Provided are therapeutic implants comprising renal tissue encapsulatedwithin a polymer bead. Also disclosed are methods for treating a diseasestate in a subject comprising implanting within said subject atherapeutic implant comprising renal tissue encapsulated within apolymer bead.

Also provided are methods for making a therapeutic implant comprising:providing renal tissue; mixing the renal tissue with a solutioncomprising a polymer, thereby forming a tissue-polymer suspension;extruding the tissue-polymer suspension into an bead-forming solution,thereby forming a therapeutic implant comprising beads of said polymerwithin which the renal tissue is encapsulated.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the results of an assessment of the relative viability ofencapsulated and non-encapsulated minced rat kidney tissue.

FIG. 2 illustrates the amount of Epo released into the culture medium,as determined on day 4 post-encapsulation by ELISA; data is included forbeads devoid of tissue (beads only), non-encapsulated, minced rat kidneytissue (tissue only), and encapsulated, minced rat kidney tissue (beadswith tissue).

FIG. 3 depicts data from a study in which the average amount of variousproteins secreted into the medium was measured after four days ofculture.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Despite the increasing interest in cell encapsulation as a method fordelivering therapeutic agents, sparse to no attention has been given tothe encapsulation of whole tissue fragments. It has presently beendiscovered that the encapsulation of minced kidney tissue provides anopportunity to deliver natural Epo and other beneficial agents fromendogenous cells, while providing an immunological barrier to preventtissue rejection. As discovered herein, the transplantation of aninducible, beneficial agent-secreting, implantable device composed ofkidney tissue can dramatically alleviate the current financial, safety,and medical issues surrounding erythropoiesis-stimulating agents. Kidneytissue encapsulation technology may also enable the development of othertherapeutic technologies for the treatment of various disease states.

In the present disclosure the singular forms “a,” “an,” and “the”include the plural reference, and reference to a particular numericalvalue includes at least that particular value, unless the contextclearly indicates otherwise. When values are expressed asapproximations, by use of the antecedent “about,” it will be understoodthat the particular value forms another embodiment. Where present, allranges are inclusive and combinable.

Provided are therapeutic implants comprising renal tissue encapsulatedwithin a polymer bead. The present implants are suitable forintroduction in vivo and for providing therapeutic effects followingimplantation. The renal tissue for use in the present implants may beautologous tissue, allogeneic tissue, xenogeneic tissue, or anycombination thereof. The renal tissue may be size-processed for use inthe present implants, for example, by mincing a source of renal tissueinto fragments. Such fragments may have a size of less than about 1 mm,or they may be larger. The size of the fragments is preferably measuredin terms of the largest dimension thereof, e.g., lengthwise if thefragments have an aspect ratio of greater than 1:1, by the length of aside if the fragments are roughly cubical, or by diameter if thefragments are roughly spherical, etc. In addition to mincing, thefragments may be further size-processed to reduce the dimensions of thetissue. For example, the fragments may be further minced so that thesize of substantially all of the fragments are less than about 300 μm,less than about 150 μm, less than about 100 μm, or less than about 50μm. The total quantity of renal tissue in an implant of the presentinvention may be at least about 100 mg, at least about 50 mg, at leastabout 30 mg, or at least about 10 mg. Various factors, such as thedesired total surface area of the renal tissue, the type of therapy, thetype of renal tissue, the characteristics of the subject undergoingtherapy, the type and stage of the disease state against which therapyis desired, the quantity and type of materials secreted by the tissue,and other factors that will be appreciated by those skilled in the art,may be used to determine the quantity of renal tissue in the implant,the size of the individual tissue fragments, or both.

The polymer bead preferably comprises a biocompatible polymer, such as anaturally occurring or synthetically derived biopolymer. The polymerbead may comprise such polymers as alginate, hyaluronic acid,carboxymethylcellulose, polyethylene glycol, dextran, agarose,poly-L-lysine, carageenan, pectin, tragacanth gum, xanthan gum, guargum, gum arabic, type I collagen, laminin, fibronectin, fibrin, or anycombination thereof. A preferred combination of polymers comprisesalginate and poly-L-lysine. Such polymers are readily commerciallyavailable.

The term “bead” when used in reference to the polymer is intended toconvey that the polymer composition generally assumes a roughlyspherical shape, but may also be ovoid or oblong. The precise shape ofthe polymer bead is not essential to the present invention; any shapethat permits the renal tissue to be substantially enveloped within thepolymer is acceptable. When measured according to its greatestdimension, a polymer bead may have a diameter of about 0.5 mm to about10 mm, and is preferably about 3 mm to about 6 mm. The size of thepolymer bead may be measured according to the characteristics of thebead prior to implantation, or following implantation. As polymer beadsmay spontaneously bud, the size of the polymer bead may be measured withrespect to an un-budded bead or with respect to a bead that results frombudding.

The characteristics of the polymer bead permit the instant implants tosecrete beneficial agents from within the bead into the ambientenvironment in which the bead is implanted or otherwise contained. Inother words, the polymer bead is permeable to substances that aresecreted by the renal tissue that is encapsulated within the bead. Therenal tissue may be endogenous, naturally occurring tissue or mayinclude cells that contain gene alterations, such as insertions of genesor portions of genes that are not naturally present. Renal tissue thatincludes gene alterations or insertions may be physically capable ofsecreting substances that endogenous or naturally occurring tissuecannot. The renal tissue and therefore in turn the implant of thepresent invention may secrete any compound that renal tissue, whetherendogenous or altered (e.g., genetically altered) is physically capableof producing. For example, the renal tissue may secrete one or morehormones, prohormones, proteins, growth factors, trophic factors, or anycombination thereof. As additional examples, and as further describedherein, the tissue may secrete one or more of erythropoietin, MCP-1,adiponectin, leptin, and MMP-2. The compounds that genetically alteredrenal tissue may secrete are theoretically virtually limitless.

Also provided are methods for treating a disease state in a subjectcomprising implanting within the subject a therapeutic implantcomprising renal tissue encapsulated within a polymer bead. Because thepresent implants are capable of secreting a number of beneficial agents,the inventive methods can be used to treat a wide variety of diseasestates. As used herein, “treatment” may refer to prophylactic therapy,or alleviation of any pathological phenotype. The disease state forwhich treatment is provided by the present invention may be anemia,stroke, cardiovascular disease, or any renal disease, i.e., anypathology that is directly or indirectly associated with improper kidneyfunction, for example, which results in improper kidney function, orwhich is caused at least in part by improper kidney function. Renaldisease may be hereditary, congenital, or acquired. Non-limitingexamples of renal disease include polycystic kidney disease, Alport'ssyndrome, hereditary nephritis, primary hyperoxaluria, cystinuria,nephritis, nephritic syndrome, hypertension, diabetes, acute kidneydisease, chronic kidney disease (persistent proteinuria), renal tubularacidosis, glomerular diseases, and Goodpasture's syndrome. The benefitsof treatment with Epo, for example has been widely documented withrespect to a number of pathologies, and is readily appreciated by thoseskilled in the art. The characteristics of the polymer beads and renaltissue for use in the present methods may be as previously describedwith respect to the inventive therapeutic implants.

The present invention is also directed to methods for making atherapeutic implant. The methods for making a therapeutic implantsuccessfully results in the fabrication of therapeutic compositions thatcan be used in accordance with the present disclosure. The presentmethods comprise providing renal tissue; mixing the renal tissue with asolution comprising a polymer, thereby forming a tissue-polymersuspension; extruding the tissue-polymer suspension into a bead-formingsolution, thereby forming a therapeutic implant comprising beads of thepolymer within which the renal tissue is encapsulated.

Renal tissue may be prepared in accordance with the previously disclosedtechniques, including selecting a tissue type and size-processing. Thepolymer solution with which the renal tissue is mixed in accordance withthe present invention may comprise a combination of a polymer and agrowth medium. The characteristics of the polymer may be determined asdescribed above. Any acceptable culture medium, nutrient broth, or thelike may be used for the instant growth medium; the characteristics ofan appropriate growth medium, which may comprise a mixture of media, arereadily determined by those skilled in the art. Growth media can vary inpH, glucose concentration, growth factors, and the presence of othernutrient components, but the growth medium should fulfill at least someof the nutritional requirements of the renal tissue, and preferablyfulfills most or all nutritional requirements, and should possess pH andother chemical characteristics necessary to sustain and nurture therenal tissue. An example of a suitable growth medium is DULBECCO'SMODIFIED EAGLES MEDIUM (DMEM; Invitrogen, Carlsbad, Calif.). Growthmedia are commercially available and suitable media are readilyrecognized by those skilled in the art. To prevent infection,antibiotics, such as penicillin, streptomycin, and the like, may beadded to the growth medium. Serum, such as fetal bovine serum, may alsobe added to the growth medium.

Mixing of the renal tissue and the polymer solution may be achieved byany means that are suitable for forming a suspension, i.e., a mixture inwhich the renal tissue is substantially uniformly suspended in thepolymer solution, such as agitation, stirring, or pouring. For example,the mixing may be achieved by loading the renal tissue and polymersolution into a first container, such as a syringe, transferring thesolution into another container, such as by expelling the contents of asyringe into a second syringe, transferring the solution back to thefirst container, and then repeating this cycle as necessary until asuspension is achieved.

After a suspension is formed, the suspension is extruded intobead-forming solution in order to form polymer beads within which therenal tissue is encapsulated. The extrusion may comprise ejecting thesuspension from a syringe, or otherwise transferring the suspension fromone container into another in which the bead-forming solution iscontained, or alternatively, transferring the bead-forming solution intoa container in which the suspension is held. The bead-forming solutionmay be ionic, may a cross-linking solution, or both. In one embodimentthe bead-forming solution comprises CaCl₂. The polymer beads withencapsulated renal tissue may form spontaneously when combined with thebead-forming solution. The process of bead-forming may be furtherassisted, for example, by agitating the mixture of the suspension andthe bead forming solution, modifying the temperature of the mixture(e.g., raising the temperature), or both.

Following the formation of the polymer beads, chemical cross-linking ofthe beads may be achieved by placing the beads into a cross-linkingsolution. For cross-linking, the beads may be transferred into a dilutesolution of polymer, preferably a different polymer than the majorpolymer component of the beads. For example, if the major component ofthe polymer bead comprises alginate, a dilution solution ofpoly-L-lysine may be used to cross-link the alginate beads. Optionally,an additional polymer layer may be added to the polymer beads followingtheir “production” in the bead-forming solution, or followingcross-linking. Preferably, the additional polymer layer comprises thesame polymer that makes up major component of the polymer beads. Forexample, an additional alginate layer may be added to a bead of whichthe major component is alginate. Adding another polymer layer to thepolymer beads may be accomplished by placing the beads in a dilutepolymer solution comprising the polymer of which the extra layer will bemade.

The present invention is further defined in the following Examples. Itshould be understood that these examples, while indicating embodimentsof the invention, are given by way of illustration only, and should notbe construed as limiting the appended claims. From the above discussionand these examples, one skilled in the art can ascertain the essentialcharacteristics of this invention, and without departing from the spiritand scope thereof, can make various changes and modifications of theinvention to adapt it to various usages and conditions.

Example 1 Formation of Therapeutic Implant

Four kidneys from female, Long Evans rats (eight weeks old) weresurgically removed, rinsed in ice cold phosphate buffered saline withoutCa²⁺ and Mg²⁺ (PBS) (Invitrogen, Carlsbad, Calif.) and then, using ascalpel, were minced into small pieces (1-5 mm³). A 300 uM-steel sieve(Sigma, St Louis, Mo.) was then used to further mince the tissuefragments. Minced tissue was then washed three times with 30-50 mL ofGrowth Medium containing Dulbecco's Modified Eagles Medium (DMEM)(Invitrogen) containing 1% penicillin/streptomycin (Invitrogen) and 1%fetal bovine serum (FBS) (Hyclone, Logan, Utah). The final wash wascompletely removed and the tissue fragments were loaded into one1-milliliter syringe of a two syringe mixing system. A 1.8% (w/v)alginate solution (Sigma) was prepared in Growth Medium and was loadedinto the second 1-milliliter syringe. The two solutions were then mixedtogether by pushing the contents back and forth through both syringes.The minced tissue-gel suspension was then extruded into a 100 mM CaCl₂solution. The resulting encapsulated tissue beads were then incubated atroom temperature in CaCl₂ with slow agitation for 5 minutes. The beadswere then chemically cross-linked by transferring into 0.05% (w/v)poly-L-lysine, molecular weight 24,000 (Sigma) containing 1% FBS for 5minutes and then coated with another layer of 0.1% (w/v) alginatesolution containing 1% FBS for 5 minutes. Four to ten beads were thentransferred to individual wells of a 24 well low-cluster, tissue culturedish containing 0.5 mL of Growth Medium, or Growth Media containing 100ng/mL poly-D-glutamic acid (pDGA) (Sigma), and cultured at 37° C. forfour days under either normoxic or hypoxic (5% Oxygen) atmosphericconditions. Beads were visually examined and imaged using a digitalcamera and Eclipse TE2000-U microscope (Nikon, Japan).

Visual examination of alginate encapsulated tissue beads showed thatmanual extrusion through the two-way syringe system was effective ingenerating spherical, tissue containing beads. Tissue fragments withinthe alginate beads were also visible, demonstrating a uniformdistribution throughout the alginate gel.

Example 2 Bead Diameter Measurements

Fourteen individual, tissue containing beads were placed into a cleantissue culture plate and imaged using a Nikon dissecting microscopefitted with a digital camera. The diameter of each bead was thenmeasured using IMAGE PRO PLUS Software.

Visual examination of alginate encapsulated tissue beads showed thatmanual extrusion through the two-way syringe system was effective ingenerating spherical, tissue containing beads. Tissue fragments withinthe alginate beads were also visible, demonstrating a uniformdistribution throughout the alginate gel. One-hundred and fifteen beadswere generated from 3.72 g of fragmented kidney tissue resulting inapproximately 32 mg of tissue per bead. Table 1 shows the distributionof bead diameter.

TABLE 1 Diameter Bead (mm) 1 4.84 2 4.26 3 4.66 4 4.13 5 4.37 6 4.13 74.83 8 4.59 9 4.36 10 4.42 11 4.34 12 5.90 13 4.55 14 4.51 Average 4.56Std 0.44The average diameter of fourteen individual beads was found to be4.56+/−0.44 mm.

Example 3 Assessment of Cell Viability

Minced kidney tissue viability was assessed using ALAMAR BLUE(Invitrogen), a colorimetric REDOX indicator that is reduced in responseto metabolic activity. After four days in culture, spent Growth Mediumwas removed from samples of non-encapsulated kidney tissue, encapsulatedkidney tissue, isopropanol fixed kidney tissue and Growth Medium only.One milliliter of Growth Medium, containing 10% ALAMAR BLUE, was addedto the samples and further incubated for 2-4 hours at 37° C., 5% CO₂with gentle rocking. Spent media was then analyzedspectrophotometrically (SPECTRAMAX-190, Molecular devices, Sunnyvale,Calif.) at 570 nm and 600 nm. Media from each sample was analyzed intriplicate. Percent reduction of ALAMAR BLUE was determined followingthe manufactures instructions and is an indirect measurement of cellviability.

After four days of culture, tissue viability was evaluated. As comparedto non-encapsulated tissue, encapsulation maintained greater tissueviability (FIG. 1). Non-encapsulated kidney tissue, cultured undereither normoxia or hypoxia showed similar relative mean viabilities of20.9%+/−3.4% and 21.0%+/−1.9%, respectively. However, encapsulatedkidney tissue, cultured under normoxic conditions showed an increase inrelative mean tissue viability. Encapsulated tissue showed a relativemean tissue viability of 83.5%+/−4.5%. Encapsulated kidney tissue,cultured under hypoxic conditions resulted in reduced tissue viabilityof 31.3%+/−3.4%. As expected, tissue fixation resulted in a significantdecrease in tissue viability to 5.0%+/−0.084%.

Example 4 Epo Secretion Analysis

After four days of culture, spent media was collected and the amount ofEpo released into the culture medium was determined using a QuantikineMouse/Rat Erythropoietin ELISA kit (R&D systems, MN). The ELISA platewas assayed spectrophotometrically (SPECTRAMAX-190, Molecular devices,Sunnyvale, Calif.) at 540 nm. Data was analyzed by comparing absorbancevalues of unknown samples to the linear regression of a standard curve.

The amount of Epo released into the culture medium was determined on day4 post-encapsulation by ELISA. Data was normalized to absorbance valuesobtained with Growth Medium only (Corrected Mean). Each measurement wasconducted on spent media obtained from 8-10 beads. Standard error of themean (SEM) was also calculated. Data shown in Table 2, below, isrepresented in graphical form in FIG. 2.

TABLE 2 MEAN SEM NORMALIZED TREATMENT GROUP (pg/mL) (pg/mL) MEAN (pg/mL)Growth Medium only −26.79 3.73 0.00 (background) Beads only −32.85 2.10−6.06 Tissue only 19.04 3.34 45.83 Beads with tissue 52.52 28.59 79.31Beads with tissue and pDGA 34.12 10.50 60.91

In FIG. 2, data bars represent the average of triplicate measurements,and error bars represent SEM. Each measurement was conducted on spentmedia obtained from 8-10 beads.

Results showed that minced kidney tissue produced 45.8+/−3.3 pg/mL ofEpo into the surrounding culture media. Likewise, alginate encapsulationdid not impede Epo release from the minced tissue, producing 79.3+/−28.6pg/mL of Epo. In order to determine if Epo production could bechemically enhanced, beads were prepared and cultured in pDGA. Resultsshowed that pDGA treatment did not effect Epo production, generating60.9+/−10.5 pg/mL of Epo. As a negative control, Epo production frombeads devoid of tissue was determined. As expected, no measurable Epowas detected from these samples.

Example 5 Trophic Factor Secretion Analysis

After four days of culture, spent culture medium was harvested from thebeads. Cell debris was removed from the spent culture medium bycentrifugation and the culture medium was stored at −80° C. At the timeof analysis, spent culture medium was assayed by ELISA for the followingprotein factors: interleukin-4 (IL-4), monocyte chemotactic protein-1(MCP-1), RANTES, granulocyte-macrophage colony stimulating factor(GMCSF), interleukin-10 (IL-10), adiponectin, leptin, matrixmetaloprotinase-2 (MMP-2) with Searchlight Proteome Arrays (PierceBiotechnology Inc.).

As compared to spent culture medium derived from beads without tissueencapsulation, beads containing kidney tissue fragments secretedelevated amounts of MCP-1 (50.6+/−8.9 pg/mL), adiponectin(132,060.6+/−11,226.7 pg/mL), leptin (10.3+/−2.6 pg/mL) and MMP-2(945.2+/−13.3 pg/mL) and low to undetectable amounts of IL-4, RANTES,GMCSF and IL-10. As shown in Table 3, below, each treatment groupcontained three samples (1, 2, 3).

TABLE 3 IL4 MCP1 RANTES GMCSF IL10 Adiponectin Leptin MMP2 pg/ml pg/mlpg/ml pg/ml pg/ml pg/ml pg/ml pg/ml Beads with tissue 1 39.8 68.4 7.6105.8 20.6 113137.8 14.6 1160.0 2 1.6 77.0 10.4 69.8 1.6 136667.8 19.61184.6 3 24.2 47.2 1.6 78.2 1.6 151719.0 10.6 1138.6 AVG 21.9 64.2 6.584.6 7.9 133841.5 14.9 1161.1 STD 19.2 15.3 4.5 18.8 11.0 19445.3 4.523.0 SEM 11.1 8.9 2.6 10.9 6.3 11226.7 2.6 13.3 Beads without tissue 132.0 14.4 1.6 63.8 13.4 1827.2 7.0 616.4 2 32.8 13.8 3.0 130.4 17.61655.2 5.1 15.6 3 32.6 12.6 1.8 118.2 18.8 1860.4 1.8 15.6 AVG 32.5 13.62.1 104.1 16.6 1780.9 4.6 215.9 STD 0.4 0.9 0.8 35.5 2.8 110.1 2.6 346.9SEM 0.2 0.5 0.4 20.5 1.6 63.6 1.5 200.3 Medium only 1 29.0 3.1 1.6 98.820.4 939.2 5.1 15.6 2 30.2 22.6 1.6 91.2 16.6 1159.0 5.1 15.6 3 34.816.8 1.6 85.0 15.0 1436.4 2.0 441.8 AVG 31.3 14.2 1.6 91.7 17.3 1178.24.1 157.7 STD 3.1 10.0 0.0 6.9 2.8 249.2 1.8 246.1 SEM 1.8 5.8 0.0 4.01.6 143.8 1.0 142.1 STD = Standard deviation, SEM = Standard error ofthe mean. Data shown here is represented in graphical form in FIG. 3, inwhich data bars represent the average amount of protein secreted intothe medium after four days of culture. Background measurements obtainedfrom beads without tissue was subtracted from the data shown. Error barsrepresent SEM.

Example 6 Evaluation of Erythropoiesis Stimulating Activity

Rat erythroid CD34+ cells (Lonza, Walkersville Md.) are resuspended at15,000 cells/cm2 in IMDM with 10% FBS. Bead conditioned medium are thenadded to methylcellulose colony forming assay medium (MethoCult GFH4534, StemCell Technologies, Vancouver BC). Cells are added to themethylcellulose and plated with subsequent incubation at 37° C., in a 5%CO2 incubator for 12-14 days. Colonies containing over 50 cells arecounted by phase contrast microscopy.

Conditioned media derived from encapsulated rat kidney tissue haspreviously been shown to contain Epo. Conditioned media is presentlyshown to have erythropoiesis stimulating activity (ESA) as measured byBFU-E activity.

Example 7 Evaluation of Renoprotective Effects of Encapsulated RenalTissue Fragments

The purpose of this study is to evaluate the renoprotective effects ofalginate encapsulated rat kidney tissue fragments in a rat model ofrenal disease. Sprague Dawley rats (diabetic or non-diabetic) with aninitial weight of 200-250 g are used for these experiments. The rats areanesthetized with an intraperitoneal injection (5 mg/kg) of a 4:1solution of ketamine hydrochloride and xylazine hydrochloride. Kidneyfailure is induced by a two-stage nephrectomy procedure. The upper andlower parts of the left kidney (two thirds of one kidney) are resectedusing silk ligature while preserving the renal capsule. Ten days later,the right kidney is removed, leaving approximately ⅙ of the total kidneymass (⅚ nephrectomy). Applying soft pressure with methylcellulose stopsbleeding, and the peritoneum and skin is closed in layers withresorbable 4-O Vicryl sutures.

Five weeks after the ⅚-nephrectomy procedure, beads are transplantedunder the capsule As a control, ⅚ nephrectomized rats are injected withfibrin matrix only. Serum samples are obtained on days 0 (prior to ⅚nephrectomy) and on day 1 (day of cell transplantation), days 7, 14, 21,28 and 35 (day of necropsy). Blood urea nitrogen and creatinine arequantified using a VETACE CHEMISTRY ANALYZER (Alpha WassermannDiagnostic Technologies, LLC, West Caldwell, N.J.).

Animals in all groups are sacrificed five weeks post celltransplantation by carbon dioxide asphyxiation. Kidneys are removed forhistology and transcriptional analysis. Half of each kidney issnap-frozen in liquid nitrogen for RT-PCR analysis. Messenger RNA isisolated from the frozen kidney tissue by study coordinator andsubjected to transcriptional analysis utilizing low-density microarraycards containing pro-fibrotic and inflammatory genes. The remainingcorneal kidney section is fixed in 10% neutral buffered formalin fordownstream histological analysis.

Kidney tissue fixed for histology will be histologically processed,sectioned (5 μm-thick) and stained with hematoxylin/eosin. Tubularinjury is evaluated and scored by a veterinary pathology.

In this study, subcapsular transplantation of alginate encapsulatedkidney tissue fragments will slow the progression of renal injury in ⅚nephrectomized rodents or in rodent models of diabetic nephropathy. Bothserum creatinine and blood urea nitrogen values are significantlyreduced in the hUTC treated animals as compared to the control animals.In addition, the bead lowers blood glucose levels in rodent models ofdiabetic nephropathy. Histological injury assessment reveals a reductionin tubular necrosis and tubular dilation in the treated animals.

Despite the increasing interest in cell encapsulation as a method fordelivering therapeutic agents, sparse to no attention has been placed onthe encapsulation of whole tissue fragments. It has herein beendemonstrated that encapsulated kidney tissue fragments secrete Epo andother beneficial agents into a culture medium. Therefore, the disclosedtherapeutic implants and therapeutic methods can provide treatment ofnumerous disease states.

The disclosures of each patent, patent application and publication citedor described in this document are hereby incorporated herein byreference, in their entirety.

1. A therapeutic implant comprising renal tissue encapsulated within apolymer bead.
 2. The therapeutic implant according to claim 1, whereinsaid renal tissue is autologous tissue, allogeneic tissue, xenogeneictissue, or any combination thereof.
 3. The therapeutic implant accordingto claim 1, wherein said renal tissue comprises genetically-alteredcells.
 4. The therapeutic implant according to claim 1, wherein saidrenal tissue comprises fragments of renal tissue, and wherein saidfragments have a size of less than about 1 mm.
 5. The therapeuticimplant according to claim 1, wherein said renal tissue comprisesfragments of renal tissue, and wherein said fragments have a size ofless than about 300 μm.
 6. The therapeutic implant according to claim 1,comprising at least about 30 mg of renal tissue.
 7. The therapeuticimplant according to claim 1, wherein said polymer bead comprisesalginate, hyaluronic acid, carboxymethylcellulose, polyethylene glycol,dextran, agarose, poly-L-lysine, carageenan, pectin, tragacanth gum,xanthan gum, guar gum, gum arabic, type I collagen, laminin,fibronectin, fibrin, or any combination thereof.
 8. The therapeuticimplant according to claim 1, wherein said polymer bead comprisesalginate and poly-L-lysine.
 9. The therapeutic implant according toclaim 1, wherein said polymer bead has a diameter of about 3 mm to about6 mm.
 10. The therapeutic implant according to claim 1, wherein saidtissue secretes one or more hormones, prohormones, proteins, growthfactors, or any combination thereof.
 11. The therapeutic implantaccording to claim 10, wherein said tissue secretes one or more oferythropoietin, MCP-1, adiponectin, leptin, and MMP-2.
 12. A method fortreating a disease state in a subject comprising implanting within saidsubject a therapeutic implant comprising renal tissue encapsulatedwithin a polymer bead.
 13. The method according to claim 12, whereinsaid disease state is anemia, stroke, cardiovascular disease, or renaldisease.
 14. The method according to claim 12, wherein said renal tissueis autologous tissue, allogeneic tissue, xenogeneic tissue, or anycombination thereof.
 15. The method according to claim 12, wherein saidrenal tissue comprises genetically-altered cells.
 16. The methodaccording to claim 12, wherein said renal tissue comprises fragments ofrenal tissue, and wherein said fragments have a size of less than about1 mm.
 17. The method according to claim 12, wherein said renal tissuecomprises fragments of renal tissue, and wherein said fragments have asize of less than about 300 μm.
 18. The method according to claim 12,wherein said therapeutic implant comprises at least about 30 mg of renaltissue.
 19. The method according to claim 12, wherein said polymer beadcomprises alginate, hyaluronic acid, carboxymethylcellulose,polyethylene glycol, dextran, agarose, poly-L-lysine, carageenan,pectin, tragacanth gum, xanthan gum, guar gum, gum arabic, type Icollagen, laminin, fibronectin, fibrin, or any combination thereof. 20.The method according to claim 12, wherein said polymer bead comprisesalginate and poly-L-lysine.
 21. The method according to claim 12,wherein said polymer bead has a diameter of about 3 mm to about 6 mm.22. The method according to claim 12, wherein said tissue secretes oneor more hormones, prohormones, proteins, growth factors, or anycombination thereof.
 23. The method according to claim 22, wherein saidtissue secretes one or more of erythropoietin, MCP-1, adiponectin,leptin, and MMP-2.
 24. A method for making a therapeutic implantcomprising: providing renal tissue; mixing said renal tissue with asolution comprising a polymer, thereby forming a tissue-polymersuspension; extruding said tissue-polymer suspension into anbead-forming solution, thereby forming a therapeutic implant comprisingbeads of said polymer within which said renal tissue is encapsulated.25. The method according to claim 24, wherein said solution comprising apolymer comprises: alginate, hyaluronic acid, carboxymethylcellulose,polyethylene glycol, dextran, agarose, poly-L-lysine, carageenan,pectin, tragacanth gum, xanthan gum, guar gum, gum arabic, type Icollagen, laminin, fibronectin, fibrin, or any combination thereof; and,a growth medium.
 26. The method according to claim 24, wherein saidbead-forming solution comprises a cross-linking solution.
 27. The methodaccording to claim 24, wherein said bead-forming solution comprises anionic solution.
 28. The method according to claim 24, wherein saidbead-forming solution comprises CaCl₂.
 29. The method according to claim28, further comprising coating said beads with an additional polymerlayer.